2014-15

Brennan Clark and Prashant Godishala - Cellular and Molecular Biology

This study shows that
lengthening the culture period for pre-transplantation adult porcine islets from
7 days to 30 days significantly lessens the chance of immunorejection after
xenotransplantation for type 1 diabetes. Immunofluorescence staining was chosen
to analyze antigen expression, islet viability, and insulin production after
culture periods of 7, 15, and 30 days. Because an inexpensive humidity chamber to
provided 95% humidity was not available, an inexpensive humidity chamber was engineered
from materials purchased at Target and found in the laboratory. Additionally, silane
gold-coated slides were optimized for immunofluorescence staining. Immunocytochemistry
staining showed that after 30 days, there was a significant reduction in Gal+
cells and in CD45+, Class I, and Class II antigens (p < 0.05),
all of which are associated with immunorejection of porcine islets. Additionally,
there was a significant increase in insulin production over a 30-day culture
period (p < 0.05), as well as a significant reduction in mononuclear cells
that are responsible for immunorejection. Furthermore, viability staining
showed that a culture period of 30 days provides a rehabilitative environment
for islets that experience stress after extraction (p < 0.05). Finally, RT-PCR
was done to validate results. RT-PCR showed no significant change in gene
expression of insulin over a 30-day culture period (p > 0.05). As a result
of our work, our research site is now running quality control trials to
determine efficacy of incubating all pre-transplantation adult porcine islets
for 30 days.

Jack Dickinson and Andrew Sit -

In this project, a state-of-the
art spaceflight fabrication tracking system was engineering that provides real-time
data to
efficiently
track and manage inventory components that are being used to manufacture
the James Webb Space Telescope and the Geostationary Operational Environmental
Satellite Series-R. Radio frequency identification (RFID) hardware was chosen
over universal
product code (UPC) barcoding and global positioning systems (GPS) based on a paper by Ong & Forgione (2013) that indicated RFID has
great potential to “improve accountability and maintenance of…aviation parts that
cost $5,000 or more,” which was our goal. Because commercial middleware
required to run RFID hardware is expensive, an inexpensive workaround was
devised by programming two layers of middleware that were integrated into a
back-end database, designed using Microsoft Access. Blender, which is open-source
three-dimensional graphics software, was used to integrate
three-dimensional modeling into the system. Furthermore, for
ease of use by Millennials, the Splashtop remote desktop application from Apple was
integrated into the systemto enable access to the database from
mobile devices. A cost-benefit analysis of the system showed a profit
margin of 180% over ten years. The system is engineered so that it can be
replicated to support large-scale and advanced aerospace programs with the goal
of expanding the use of RFID technology in the aerospace industry.

Siddarth Eswarachari and Moira Southern - Bioinformatics

Carcinogenic aromatic
hydrocarbons are being released into water supplies in increasing
concentrations each year. One technique tobioremediate these carcinogens is to bioengineer bacteria to catabolize
(degrade) the aromatic hydrocarbons. The bacteria Pseudomonas putida F1 shows promise in bioremediation of aromatic
hydrocarbons; however, before P. putida
F1 can be bioengineered to catabolize aromatic hydrocarbons, ways in which P. putida F1 catabolize “more
appetizing” aromatic nutrients, such as homogentisate, must be found. We
focused on the homogentisate catabolic pathway because this pathway is the
central route into which the aromatic amino acids L-phenylalanine and
L-tyrosine funnel in the bacteria P.
putida Uand P. putida KT2440, which are closely related to P. putida F1. Using the online databases
FASTA in GenBank, BLAST, T-COFFEE, and WebLogos, we compared protein sequences
for P. putida F1 to other closely
related bacterial strains that included P.
putida KT2440, P. aeruginosa PAO1,
P. fluorescens Pf0-1, and Comamonas testosterone CNB-1 to identify
substrates and enzymes in the pathway by which homogentisate is catabolized by P. putida F1. We then used TMHMM and PSORTb to determine the transporter and regulatory
proteins for the homogentisate pathway in P.
putida F1. E-values were used to validate best-fit protein sequences,
showing 100% accuracy for choices of enzymes identified for the homogentisate
pathway in P. putida F1.
Consequently, our results were published on GENI-ACT, which is a database that
contains genomic information on 2775 bacteria.

We investigated
mechanisms by which organic triblock copolymers can serve as molecular
band-aids in sealing cardiac muscle-cell membranes that have been damaged by
Duchenne muscular dystrophy. Molecular dynamics simulations were carried out on
copolymers and their hydrophilic and hydrophobic subunits at varying physical
and chemical compositions to examine effects of molecular structure and
temperature on the mechanism by which the copolymers insert into healthy,
partially damaged, and fully damaged muscle-cell membranes. The collapse or
expansion of the copolymers was monitored by examining the radius of gyration
and solvent accessible surface area of copolymers during the insertion
processes, a technique that is novel to the field.

Results showed
that copolymers significantly increase in size after insertion into damaged
membranes (p < 0.05), and elevated temperatures significantly enhance
insertion(p < 0.05). Additionally,
short-chained triblock copolymers show the most potential in repairing
partially damaged membranes while larger triblock hydrophobic copolymers are
most effective in repairing fully damaged membranes. This suggests that
short-chain triblock copolymers are most effective in early stages of muscular
dystrophy while longer-chain triblock copolymers are most effective in the
later more severe stages of muscular dystrophy. Results also suggest that
hydrophobic subunits of triblock copolymers are the driving force behind
insertion while the hydrophilic subunits cause the expansion of the copolymers
within damaged membranes. Furthermore, none of the copolymers studied inserted into undamaged membranes at any
temperatures simulated, suggesting that the copolymers may safely serve as molecular
bandages without affecting undamaged tissue.

Sofie Kim and Jake Levy - Chemistry

Because of its ability to serve as a
linkage within larger molecules, 1,2,3-triazole derivatives are widely used in pharmaceuticals
that require linkages to facilitate stability, utility, and permeability. This
study reports the first documented synthesis and optimization of the 4,5-
(bis)trimethylsilyl-5-ethynyl-1,2,3-triazole; the 5-ethynyl-1,2,3-triazole; and
the 4-trimethylsilyl- 5-ethynyl-1,2,3-triazole, using environmentally friendly
and cost-effective synthesis protocols.

Synthesis of the 4,5-(bis)trimethylsilyl-5-ethynyl-1,2,3-triazole
involved a 1,3-dipolar cycloaddition reaction between benzyl azide and 1,4(bistrimethylsilyl)-1,3-butadiyne
(BTMSBD). Optimization of this reaction, which involved testing varying
quantities of BTMSBD, which gave a maximized yield of 82%. This synthesis was
followed by a silver tetrafluoraborate-, copper(II) sulfate-, and sodium
L-ascorbate(+)-catalyzed desilylation of the
4,5-(bis)trimethylsilyl-5-ethynyl-1,2,3-triaozle to synthesize the 5-ethynyl-1,2,3-triazole.
Optimization of the 5-ethynyl-1,2,3-triazole involved varying time,
temperature, and reagent quantities, and gave a maximized yield of 73%. During
optimization of the 5-ethynyl-1,2,3-triazole, a less expensive silver ion
catalyst, silver nitrate, was tested, which resulted in the unexpected
synthesis of 4-trimethylsilyl-5-ethynyl-1,2,3-triazole. The 4-trimethylsilyl-5-ethynyl-1,2,3-triazole
was optimized in terms of reagent quantities, time, and temperature to give a
maximized yield of 92%. These
results are important in the context of chemoselectivity, illuminating that
small differences in time, temperature, and amounts and choice of reagents can lead
to significant distinctions in the final product. The optimized protocols developed for the
three 5-substituted-1,2,3-triazoles reported in this paper will be published
and then added to a chemical library to enable syntheses of additional 1,2,3-triazole
derivatives for use in pharmaceutical drug discovery.

Grace Kirkpatrick and Annie McFarland - Biology

This study reports significant damage to dendrites (neural information receivers in synapses) in the CA1 stratum radiatum region of the hippocampal due to hyperglycemia. Hyperglycemia is a disorder caused by abnormally high blood glucose levels (> 125 mg/dL) that puts 80% of extremely preterm babies born each year at risk for decreased brain mass, intraventricular hemorrhage, blindness, and possible damage to the hippocampus, which is the area of the brain responsible for memory and spatial reasoning (1).

Fluorescent immunohistochemistry was selected to stain for greater sensitivity of dendrite visualization in tissues of the CA1 stratum radiatum region of the hippocampus from a hyperglycemic rat model. When some tissues stained poorly with the primary antibody anti-beta tubulin, chosen for its structural component of microtubules and critical role in axon growth and maintenance, a small-scale test was developed using the primary antibody microtubule associated protein-2 (MAP2) to isolate the staining problem. Results suggested that MAP2 was more effective in staining. Then, Adobe Photoshop CS6 image analysis was pioneered in the lab to quantify fluorescence from stained dendrites by fluorescence of tagged dendrites in pixesl, rather than numbers of dendrites expressed relative to number of neurons (i.e. dendrites/neuron). To eliminate bias, Photoshop CS6 analyses were blinded. Adobe Photoshop CS6 worked well, effectively eliminating the need to manually count dendrites, thus decreasing error and time needed for analysis. Results showed significant damage to dendrites in tissues of the CA1 stratum radiatum region of the hippocampus due to hyperglycemia (p = 0.024).

Rustam Kosherbay - Computer Science

The purpose of this study was to program
a flight simulator with a virtual drone that generates energy-efficient flight
paths over farm fields to monitor agricultural crops. 3D models of the virtual drone
and a virtual agricultural field were developed in Blender using low-polygon 3D
modeling style to minimize random-access memory (RAM) use and were then imported
into Unity. A navigational algorithm for the virtual drone was developed employing
Monte Carlo methods, specifically simulated annealing.
Mathematical functions were developed to achieve maximal field coverage with minimal
energy consumption.Results demonstrated that the
distance-traveled and area-of-vision functions operate simultaneously to
identify flight paths with minimal energy costs and maximum coverage over
agricultural fields. The navigational algorithm works to minimize energy spent
by the drone by modifying the flight path such that the ideal score of the flight
path approaches zero. By randomizing the initial flight paths using Monte-Carlo
methods, the algorithm improves
progressively as it operates.

Tests of the system showed that
the sinusoidal patterns farmers typically use to plant and harvest crops are
significantly less effective than logarithmic spiral patterns or computer-randomized
patterns (p < 0.05). However, there was no significant difference among the
flight paths that began with logarithmic spirals or computer randomized paths
(p > 0.05). The most efficient flight path overall was achieved with a
flight pattern that was initially computer randomized with a-value of 0.7, which gives 70% priority to
distance-traveled and 30% priority to area-of-vision.

Studies by Lee et
al. (2008) and Park et al. (2007) formed the basis of our study. Lee et al.
reported that crystalline organic semiconducting materials have higher electron
mobility than amorphous organic semiconductors that are currently used in
organic electronics. Based on the study by Park et al. (2007) that showed organic
semiconducting anthracene has only one accessible crystal structure at room
temperature and pressure, we choose to experiment with anthracene since we did not
have the capability to differentiate between polymorphs using a polarized
microscope.

For our methods, we
chose to use melt-process crystallization to engineer anthracene crystals based
on surface crystallization techniques used by Wu & Yu (2006) to crystallize
the organic semiconductor indomethacin. We varied cooling rates of the molten
anthracene based on work by Manoury et al. (2007) that studied cooling rates to
crystallize 5,5’-bis(4-hexylphenyl)-2,2’-bithiophene, which is not a
semiconductor. Finally, based on a study by Kéna-Cohen, Davanço, & Forrest (2008) that developed
anthracene crystals using melt processing with a gold-plated capillary-force
apparatus, we decided to employ a simpler, less expensive apparatus to
incorporate capillary force into the melt-crystallization process.

Dr. R. Holmes
served as our mentor. N. Morgan and T. Fielitz provided training on the
ultraviolet-visible spectrometry and X-ray diffraction processes we used to
analyze crystals, the sonication process that we used to clean substrates, and
the polarized optical microscope that we used to analyze crystals. In addition,
Ms. L. Fruen and the Breck Science Research team gave suggestions on our paper
and guidance throughout the extent of our study. The University of Minnesota
Department of Chemical Engineering and Materials Science provided funding,
materials, and equipment for our work.

Evelyn McChesney and Maddy McCue - Biomedical Engineering

Three avirulent strains of bacteria
were engineered to produce antimicrobial peptides (AMPs) that are capable of
replacing antibiotics in treating bacterial infections. Two major problems associated with using AMPs were addressed.
First, because AMPs are broken down by enzymes in the stomach, enzyme-resistant
probiotic bacteria were engineered as delivery systems. Second, the two main
categories of AMPs—gram-positive bacteriocins and gram-negative
microcins—require different delivery systems. The gram-positive bacteria
Lactococcus lactis IL1403 was chosen as a delivery system for bacteriocin
Abp118 because it is an AMP that has activity against gram-positive pathogens.
The gram-negative bacteria Escherichia coli MC4100 and E. coli
BL21 were chosen as delivery systems for microcin V and microcin J25 they are
AMPs that have activity against gram-negative pathogens.

AMP-producing bacteria grew in chemically
simulated conditions that mimicked the gastrointestinal tract.
These results suggest that engineered bacteria
show promise as alternatives to conventional antibiotics in targeting
pathogens.

Luke Onopa - Water Pollution and Water Quality

Triclosan, an antibiotic found in hand disinfectants,
and perfluorooctane sulfonate, an organofluorine compound found in food
wrappers and textiles, are entering wastewater in increasingly disturbing
concentrations. This study showed that a combination of these pollutants significantly
disrupts methanogens (Methanopyrus
kandleri), which are anaerobic digesters that play a critical role in
anaerobic wastewater treatment. Methods used include populating methanogens in
the presence of triclosan with and without perfluorooctane sulfonates. A simple
water-displacement method was developed to measure total methane (biogas)
production by methanogens over the course of four weeks. Results showed that
low concentrations of the antibiotic triclosan alone do not disrupt function of
methanogens (p > 0.05), while high
concentrations of triclosan do significantly disrupt methanogens (p < 0.05). Additionally, when methanogens
were in an environment with perfluorooctane sulfonate alone, the methanogen
function was not significantly disrupted (p
< 0.05). However, methanogen
function was significantly disrupted in an environment of perfluorooctane
sulfonate mixed with a high concentration of triclosan (p < 0.05), as well when in an environment of perfluorooctane
sulfonate mixed with a low concentration of triclosan (p < 0.05), suggesting that perfluorooctane sulfonate has a
significant biomagnification effect on triclosan. These results suggest that triclosan
and perfluorooctane sulfonate that are entering wastewater in increasingly disturbing
numbers may significantly disrupt anaerobic digesters that play an
indispensable role in anaerobic wastewater treatment. This has critical
implications on the future ecology of lakes and rivers as well as on the safety
of drinking supplies.

Karesten Salveson and Eve Zelickson -

The purpose of our study was to investigate the affinity of
zebra mussels (Dreissena
polymorpha) towards fresh water macrophytes and
determine locations on the macrophytes where the mussels prefer to settle.
Samples of three macrophytes—coontail (Ceratophyllum demersum), Eurasian
watermilfoil (Myriophyllum spicatum),
and clasping leaf pondweed (Potamogeton richardsonii)—were sampled via scuba diving from three different
bays (Gray’s Bay, Cook’s Bay, and North Arm) in Lake Minnetonka, MN, over the
course of 12 weeks during the summer of 2014. The mussel density (mussels/g dry
mass) was determined for each species of macrophytes as well as on each section
(top, bottom, and middle) of the macrophytes. Zebra mussels showed significant
preference for Eurasian watermilfoil compared to coontail and clasping leaf pondweed
(p < 0.05). Preference for the top
section of the macrophytes was significantly less than for the bottom and
middle sections (p < 0.05). Our
study is novel in the field of D. polymorpha biology.

Grant Two Bulls -

This study examined impacts of an early 19th
century Mdewakanton Dakota settlement on the ecology of Lake Calhoun in
Minneapolis, MN. Lacustrine coring was
used to take two core samples from Lake Calhoun at 44.94362 latitude and -93.31303 longitude and at 44.94192 latitude and
-93.31292 longitude, each representing over 400 years of sediment
accumulation. Because there were no funds available for Pb-210 dating of the cores, a
technique called loss-on-ignition
dating was run using a prototcol outlined by Engstrom, Balogh, & Swain (2007). By taking core samples at the same longitude and latitude in
Lake Calhoun as Engstrom did, it was possible to compare loss-on-ignition results toloss-on-ignition/lead-210 data reported by Engstrom to date the
cores.

Pollen present in the cores was counted based on
work by McLauchlan (2003) that showed pollen counts can be employed to determine quantitative measures of the prevalence of different
types of plants in a core sample.Pollen counts showed that the
shoreline around Lake
Calhoun was predominately
oak at the time of the settlement (AD 1830-40), with no significant
change in oak pollen in lake sediments over that period of time (p = 0.096 ). However, results did show a significant increase in ragweed (p = 0.039) and grass pollen,
which includes corn, (p = 0.040) at the time of the settlement compared to
dates before the settlement.

This study succeeded in using
two highly reproducible methods for examining historical lake ecology over time
that may prove useful in future studies. Pollen types were identified in
sediment from Lake Calhoun, and the loss-on-ignition data
matched well to data reported in the study by Engstrom, Balogh, & Swain (1).